1. Field of the Invention
The invention relates to convenient and efficient methods of assay and diagnosis of metabolic states, particularly gluconeogenesis, pyruvate flux and anapleurosis.
2. Description of Related Art
Current methods for the measurement of gluconeogenesis in humans require exposure to radioactive materials. Unfortunately, this feature precludes almost any quantitative studies of these pathways in patients. The development of .sup.13 C NMR for clinical applications is very attractive because of its convenience and the dramatic improvement in metabolic detail which it can provide. Although direct in vivo NMR spectroscopy is the most exciting application for .sup.13 C tracer studies, it is unlikely that appropriate whole-body systems will be widely available any time soon for clinical research. On the other hand, analytical NMR spectrometers suitable for .sup.13 C NMR studies of human blood or urine are already in place in every medical school in this country.
Measurement of hepatic gluconeogenesis using both .sup.13 C and .sup.14 C-labeled glucose is by now a well-developed approach and had been successfully applied in both animals and humans. However, measurements of absolute anaplerotic fluxes have been largely restricted to perfused organs and tissues and the methods cannot be performed in vivo. Since only gluconeogenesis can currently be measured noninvasively, a necessary simplification is to assume that gluconeogenesis is equal to anapleurosis. The penalty for this assumption is that the total anaplerotic flux is underestimated to an unknown degree, and the allocation of anaplerotic carbons between gluconeogenesis and the other biosynthetic pathways is not known.
There is widespread interest in measurement of flux through gluconeogenesis in animals and humans with the goal of a better understanding of dietary and hormonal regulation of fluxes through all associated pathways. Numerous techniques have been used for such measurements, including GC-mass spectrometry (Katz et al., 1993; Katz, 1985; Tayek and Katz, 1996) NMR (Cohen, 1981; Cohen, 1987; Cohen, et al. 1981), and radiotracers (Strisower et al., 1952; Landau et al., 1993). One key control point that connects these two pathways is the interconversion of PEP and pyruvate, catalyzed in the forward direction by pyruvate kinase and in the reverse direction by the combined action of pyruvate carboxylase and phosphoenolpyruvate carboxykinase. Many early studies of gluconeogenesis detected excess cycling of three carbon units through this metabolic intersection. This process, often referred to as pyruvate recycling on an excess substrate cycle, has been detected in studies of isolated hepatocytes (Clark et al., 1973; Jones and Titheradge, 1996), perfused livers (Friedman et al., 1971), and in rats and humans (Magnusson et al., 1990; Petersen et al., 1994).
Most early metabolic models have relied on measurements of isotope enrichment in two or more sites of various product molecules. In prior metabolic studies of pathways associated with the Krebs citric acid cycle, the detection of .sup.13 C-.sup.13 C spin-spin NMR couplings in product molecules has been demonstrated. This approach, which is referred to as .sup.13 C isotopomer analysis, can in some cases provide more information about isotope labeling patterns than other tracer methods. Recently, the analysis of propionate metabolism in the isolated heart under conditions where the pathway succinyl-CoA.fwdarw.pyruvate.fwdarw.acetyl-CoA was significant (Jeffrey et al., 1996) was reported. In contrast to entry of labeled acetyl-CoA and scrambling of this label in cycle reactions, this metabolic condition yields a series of nonlinear equations, which in general are difficult to solve analytically. Although the Newton-Raphson procedure is well known (Press et al., 1988), the complexity of the equations in the comprehensive model obscures some simple and helpful relations between the .sup.13 C NMR spectrum and metabolic state. One such condition occurs during hepatic metabolism of [1,2,3-.sup.13 C]propionate.